Abstract: Objective: high performance capillary electrophoresis (HPCE) Determination of Glycyrrhiza uralensis Fisch in acid. Method: buffer 3Omol/L borate solution, pH=9.2, uncoated elastic quartz capillary (75μmX 47.5cm, effective separation length 40cm) as separation channel, pressure injection (250 Kpa·S), 20kV constant pressure electrophoresis (25 ° C) separation, 254 nm detection. Results The whole analysis process can be completed within 7 min, and the linear range of glycyrrhizic acid content is 0-500 μg/ml. (r=0.999 7). The retention time of glycyrrhizic acid and the RSD value of the integrated peak area of ​​glycyrrhizic acid were 0.2% and 3.4%, respectively. Conclusion: This method is simple, rapid and reproducible. It is suitable for the rapid determination of glycyrrhizic acid in licorice, and it also provides a feasible basis for further establishment of fingerprint of licorice. Moreover, the hydrolysis product of glycyrrhizic acid, glycyrrhetinic acid, can be simultaneously analyzed, which can be used to further study the metabolism of glycyrrhizae drugs.
Key words: capillary electrophoresis, licorice, glycyrrhizic acid, glycyrrhetinic acid, fingerprints, licorice is the dry rhizome of Gscyrrhizauralensis Fisch., a perennial herb of the leguminous family, with a sweet taste and into the twelve meridians. The effect of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, moistening the lungs and relieving cough, relieving pain, and reconciling various medicines. For spleen and stomach weakness, fatigue, fatigue, palpitations, shortness of breath, cough, wrist and abdomen, limbs, acute pain, swollen sore, relieve drug toxicity, potency. The main active ingredient of licorice is glycyrrhizic acid. It has been proven to have good effects on cardiovascular, respiratory, digestive and endocrine diseases, and has anti-inflammatory, anti-allergic, anti-oxidative and anti-allergic effects. Detoxification, anti-pathogenic microorganisms and anti-cancer effects, Zui Jin, German scholar J Cinatl et al reported that glycyrrhizic acid has a certain effect on the treatment of SARS. The active ingredient of the detoxification effect of licorice is glycyrrhizic acid. After hydrolysis of one molecule of glycyrrhizic acid, one molecule of glycyrrhetinic acid and two molecules of glucuronic acid are produced, which are combined with the poison containing hydroxyl group to detoxify. At present, high-performance liquid chromatography ( HPLC ) is often used for the study of chemical constituents in medicinal herbs, but for some substances that retain very strong on the column, such as glycyrrhetinic acid, the separation time is quite long and cannot be analyzed by HPLC. High-performance capillary electrophoresis (HPCE) combines the dual separation characteristics of electrophoresis and chromatography. The information reflected by the spectrum is more detailed and comprehensive than the liquid chromatogram. It can simultaneously detect glycyrrhizic acid and its hydrolyzate when analyzing licorice drugs. Glycyrrhetinic acid, the analysis process does not exceed 7min. In this paper, a high performance capillary electrophoresis method was established, which can simultaneously separate the main chemical components of licorice, glycyrrhizic acid and its hydrolysate, glycyrrhetinic acid, and compare the difference of glycyrrhizic acid content in 7 different kinds of medicinal herbs. The method is simple, rapid and heavy. It is currently satisfactory and is generally applicable to the pharmacological research of licorice drugs.
1 Instruments and reagent instruments: HP3DCE capillary electrophoresis system (HP company, USA), DAD detector; uncoated elastic quartz capillary column (Hebei Yongnian Optical Fiber Factory); Orion 828 pH tester (Orion, USA); HD0208 ultrasonic instrument "Li kitchen" multi-function food pulverizer; Milli-Q ultra-pure water device.
Reagents: monoammonium glycyrrhizinate and glycyrrhetinic acid chemical reference (provided by China National Institute for the Control of Pharmaceutical and Biological Products); borax, sodium hydroxide (analytical grade, Shanghai Chemical Reagent Company); ultrapure water; medicinal herbs from Inner Mongolia Yili Company provide.
2 Electrophoresis conditions: 30mmol/L borate (pH 9.2) buffer as electrophoresis medium, uncoated elastic quartz capillary (75μm × 50cm, effective separation length 42.2cm) as separation channel, pressure injection (250Kpa·S), 20 kV constant pressure electrophoresis (25 ° C) separation, 254 nm detection. Before the experiment, the buffer solution was degassed by ultrasonic for 10 min, and the capillary was washed with H2O, 0.1 mol/L NaOH, 1 mol/L NaOH, 0.1 mol/L NaOH and H2O for 10 min, 10 min, 15 min, 10 min, respectively. 10 min, rinse with electrophoresis medium for 3 min before each electrophoresis.
3 solution configuration
3.1 Standard solution configuration Weigh accurately 0.050g monoammonium glycyrrhizinate, use water as solvent, and make up to 50mL volumetric flask as the reference solution of monoammonium glycyrrhizinate (1000μg/mL). Accurately weigh 0.050 g of glycyrrhetinic acid, and use ethanol as solvent to make a volume of glycyrrhetinic acid reference stock (1000 μg/mL) in a 50 mL volumetric flask.
3.2 Preparation of sample solution Sample source: “Liangwai†licorice GAP base in Erdos, Inner Mongolia; harvesting time: March 2002; extraction method: According to the results of previous experiments in this laboratory, ultrasonic extraction was used to obtain good extraction efficiency of zui; Extraction step: accurately weigh 1.00 g of licorice samples, configure ethanol-water-ammonia (10:90:0.5) extract, extract with ultrasonic for 30 min, and make up to 50 mL. Take 1 mL of the extract into a centrifuge tube, centrifuge at high speed (10 000 rp), and centrifuge the supernatant after centrifugation for testing.
4 Results and discussion
4.1 Linear relationship The standard solution of glycyrrhizic acid monoamine salt at 0, 100, 300, 500 μg/mL was prepared and injected. The concentration of the monoammonium glycyrrhizinate standard X (μg/mL) was plotted on the abscissa, and the integrated peak area value Y was plotted on the ordinate. The regression equation was calculated as Y=0.4177X+0.578 7, (r=0.9997). The results showed that the signal of the sample showed a good linear relationship with the increase of concentration in the range of 0-500 μg/mL.
4.2 Precision Experiment A sample with the batch number Rg-X-002 was taken, and the sample was prepared according to the above preparation method, and the sample was continuously injected 6 times under the selected conditions.
4.3 Determination of licorice samples The experiment compared the differences in glycyrrhizic acid content among seven different types of licorice.
4.4 Discussion In this experiment, the main chemical components of the medicinal herbs were separated by high performance capillary electrophoresis. The whole separation process was completed within 7 minutes. The content of glycyrrhizic acid in seven different kinds of licorice was determined by this method. The results showed that the content of glycyrrhizic acid in seven kinds of licorice was in the order of wild (Grade B) > Wild (Grade C) > Artificial (Grade B) ) > Artificial (Grade C) 2 years > Artificial (Grade C) 3 years; Wild > Artificial cultivation; Under the same growth conditions, Grade B > Grade C. This result is consistent with the established grade classification.
Compared with the HPLC method, the experimental method is more concise and rapid. However, the reproducibility of the HPCE method is inferior to that of the HPLC method, which makes the application of HPCE in establishing the fingerprint of Chinese herbal medicines limited. However, if the retention value is changed to the relative retention value (retention time is changed to relative retention time; peak area is changed to the peak area ratio of the internal standard), the disadvantages of the HPCE method can be overcome, and the performance of HPCE is not comparable to HPLC. up and down. The next step will focus on the development of HPCE fingerprinting technology for licorice drugs.
references
[1] SUN Xiuying (Sun Xiuying), HUANG Lijuan (Huang Lijuan), LIU Xia (Liu Xia). Analysis of the chemical components in Glyeyrrhizauralensis Fisch. Acta Chin Medand Phar (Journal of Traditional Chinese Medicine). 1994, 5:40.
Key words: capillary electrophoresis, licorice, glycyrrhizic acid, glycyrrhetinic acid, fingerprints, licorice is the dry rhizome of Gscyrrhizauralensis Fisch., a perennial herb of the leguminous family, with a sweet taste and into the twelve meridians. The effect of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, moistening the lungs and relieving cough, relieving pain, and reconciling various medicines. For spleen and stomach weakness, fatigue, fatigue, palpitations, shortness of breath, cough, wrist and abdomen, limbs, acute pain, swollen sore, relieve drug toxicity, potency. The main active ingredient of licorice is glycyrrhizic acid. It has been proven to have good effects on cardiovascular, respiratory, digestive and endocrine diseases, and has anti-inflammatory, anti-allergic, anti-oxidative and anti-allergic effects. Detoxification, anti-pathogenic microorganisms and anti-cancer effects, Zui Jin, German scholar J Cinatl et al reported that glycyrrhizic acid has a certain effect on the treatment of SARS. The active ingredient of the detoxification effect of licorice is glycyrrhizic acid. After hydrolysis of one molecule of glycyrrhizic acid, one molecule of glycyrrhetinic acid and two molecules of glucuronic acid are produced, which are combined with the poison containing hydroxyl group to detoxify. At present, high-performance liquid chromatography ( HPLC ) is often used for the study of chemical constituents in medicinal herbs, but for some substances that retain very strong on the column, such as glycyrrhetinic acid, the separation time is quite long and cannot be analyzed by HPLC. High-performance capillary electrophoresis (HPCE) combines the dual separation characteristics of electrophoresis and chromatography. The information reflected by the spectrum is more detailed and comprehensive than the liquid chromatogram. It can simultaneously detect glycyrrhizic acid and its hydrolyzate when analyzing licorice drugs. Glycyrrhetinic acid, the analysis process does not exceed 7min. In this paper, a high performance capillary electrophoresis method was established, which can simultaneously separate the main chemical components of licorice, glycyrrhizic acid and its hydrolysate, glycyrrhetinic acid, and compare the difference of glycyrrhizic acid content in 7 different kinds of medicinal herbs. The method is simple, rapid and heavy. It is currently satisfactory and is generally applicable to the pharmacological research of licorice drugs.
1 Instruments and reagent instruments: HP3DCE capillary electrophoresis system (HP company, USA), DAD detector; uncoated elastic quartz capillary column (Hebei Yongnian Optical Fiber Factory); Orion 828 pH tester (Orion, USA); HD0208 ultrasonic instrument "Li kitchen" multi-function food pulverizer; Milli-Q ultra-pure water device.
Reagents: monoammonium glycyrrhizinate and glycyrrhetinic acid chemical reference (provided by China National Institute for the Control of Pharmaceutical and Biological Products); borax, sodium hydroxide (analytical grade, Shanghai Chemical Reagent Company); ultrapure water; medicinal herbs from Inner Mongolia Yili Company provide.
2 Electrophoresis conditions: 30mmol/L borate (pH 9.2) buffer as electrophoresis medium, uncoated elastic quartz capillary (75μm × 50cm, effective separation length 42.2cm) as separation channel, pressure injection (250Kpa·S), 20 kV constant pressure electrophoresis (25 ° C) separation, 254 nm detection. Before the experiment, the buffer solution was degassed by ultrasonic for 10 min, and the capillary was washed with H2O, 0.1 mol/L NaOH, 1 mol/L NaOH, 0.1 mol/L NaOH and H2O for 10 min, 10 min, 15 min, 10 min, respectively. 10 min, rinse with electrophoresis medium for 3 min before each electrophoresis.
3 solution configuration
3.1 Standard solution configuration Weigh accurately 0.050g monoammonium glycyrrhizinate, use water as solvent, and make up to 50mL volumetric flask as the reference solution of monoammonium glycyrrhizinate (1000μg/mL). Accurately weigh 0.050 g of glycyrrhetinic acid, and use ethanol as solvent to make a volume of glycyrrhetinic acid reference stock (1000 μg/mL) in a 50 mL volumetric flask.
3.2 Preparation of sample solution Sample source: “Liangwai†licorice GAP base in Erdos, Inner Mongolia; harvesting time: March 2002; extraction method: According to the results of previous experiments in this laboratory, ultrasonic extraction was used to obtain good extraction efficiency of zui; Extraction step: accurately weigh 1.00 g of licorice samples, configure ethanol-water-ammonia (10:90:0.5) extract, extract with ultrasonic for 30 min, and make up to 50 mL. Take 1 mL of the extract into a centrifuge tube, centrifuge at high speed (10 000 rp), and centrifuge the supernatant after centrifugation for testing.
4 Results and discussion
4.1 Linear relationship The standard solution of glycyrrhizic acid monoamine salt at 0, 100, 300, 500 μg/mL was prepared and injected. The concentration of the monoammonium glycyrrhizinate standard X (μg/mL) was plotted on the abscissa, and the integrated peak area value Y was plotted on the ordinate. The regression equation was calculated as Y=0.4177X+0.578 7, (r=0.9997). The results showed that the signal of the sample showed a good linear relationship with the increase of concentration in the range of 0-500 μg/mL.
4.2 Precision Experiment A sample with the batch number Rg-X-002 was taken, and the sample was prepared according to the above preparation method, and the sample was continuously injected 6 times under the selected conditions.
4.3 Determination of licorice samples The experiment compared the differences in glycyrrhizic acid content among seven different types of licorice.
4.4 Discussion In this experiment, the main chemical components of the medicinal herbs were separated by high performance capillary electrophoresis. The whole separation process was completed within 7 minutes. The content of glycyrrhizic acid in seven different kinds of licorice was determined by this method. The results showed that the content of glycyrrhizic acid in seven kinds of licorice was in the order of wild (Grade B) > Wild (Grade C) > Artificial (Grade B) ) > Artificial (Grade C) 2 years > Artificial (Grade C) 3 years; Wild > Artificial cultivation; Under the same growth conditions, Grade B > Grade C. This result is consistent with the established grade classification.
Compared with the HPLC method, the experimental method is more concise and rapid. However, the reproducibility of the HPCE method is inferior to that of the HPLC method, which makes the application of HPCE in establishing the fingerprint of Chinese herbal medicines limited. However, if the retention value is changed to the relative retention value (retention time is changed to relative retention time; peak area is changed to the peak area ratio of the internal standard), the disadvantages of the HPCE method can be overcome, and the performance of HPCE is not comparable to HPLC. up and down. The next step will focus on the development of HPCE fingerprinting technology for licorice drugs.
references
[1] SUN Xiuying (Sun Xiuying), HUANG Lijuan (Huang Lijuan), LIU Xia (Liu Xia). Analysis of the chemical components in Glyeyrrhizauralensis Fisch. Acta Chin Medand Phar (Journal of Traditional Chinese Medicine). 1994, 5:40.
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